Procedures for detecting and identifying infectious organisms are some of the most critical tasks performed in the clinical laboratory. Whereas laboratory diagnoses of infectious diseases formerly were made by experienced microbiologists using visual inspection of stained clinical material, more rapid and objective results are obtainable using modern techniques. Immunoassays, including radioimmunoassays, enzyme-linked immunoassays, and latex agglutination and immunoblotting assays have developed into powerful diagnostic tools having utilities that are enhanced by the availability of monoclonal antibodies. Nucleic acid hybridization assays have been developed to detect microorganisms, and more recent advances in signal and target amplification have introduced the era of molecular diagnostics based on the use of oligonucleotide probes. Generally, a probe is a single-stranded polynucleotide having some degree of complementarity with a nucleic acid sequence that is to be detected (“target sequence”). A double-stranded nucleic acid hybrid between the probe and the target sequence results if the target sequence is contacted under hybridization-promoting conditions with a probe having a sufficient number of contiguous bases complementary to the target sequence. DNA/DNA, RNA/DNA or RNA/RNA hybrids may thus be formed under appropriate conditions. Probes commonly are labeled with a detectable moiety such as a radioisotope, a ligand, or a colorimetric, fluorometric or chemiluminescent moiety to facilitate the detection of hybrids.
Indeed, clinical microbiologists now use an extensive array of techniques for identifying infectious organisms (see Manual of Clinical Microbiology Murray et al., eds., 6th edition, ASM Press (1995)). Automated substrate utilization systems typically rely on enzymatic reactions that release chromogenic or fluorogenic compounds, tetrazolium-based indicators of metabolic activity in the presence of different carbon sources, or detection of the acid products of metabolism. The patterns of positive and negative reactions with these substrates establish a biochemical profile that can be used to identify microorganisms isolated from clinical samples. The chromatographic profiles of the more than 300 fatty acids that contribute to the formation of lipids in bacteria and yeast have also been used to phenotype microorganisms. Despite the availability of these very powerful techniques, polynucleotide-based assays are rapidly gaining popularity in clinical laboratory practice.
The specificity of polynucleotide hybridization reactions, together with the extraordinary sensitivity afforded by nucleic acid amplification techniques, has made molecular diagnostics the method of choice for detecting and identifying microbes that are available in only very small quantities. Commonly used DNA probe hybridization formats include: solid phase hybridization, solution-phase hybridization and in situ hybridization. In solid phase hybridization methods, a sample containing microbial polynucleotides is immobilized to a solid support, denatured and then probed with a polynucleotide probe that harbors a detectable label. Unhybridized probe is removed from the system and specifically hybridized probe detected, for example, by autoradiography or direct visual observation. In solution-phase hybridization procedures, the target polynucleotide and the labeled probe are free to interact in an aqueous hybridization buffer. Specifically hybridized probe is then detected as an indicator of the presence of target polynucleotides in the mixture. In situ hybridization using formalin-fixed tissue sections is used for obtaining information about the physical distribution and abundance of microorganisms.
One example of an organism for which a number of polynucleotide-based assays have been described is Staphylococcus aureus. For example, Milliman in U.S. Pat. No. 5,292,874 describes a hybridization-based assay to distinguish S. aureus from other Staphylococcus spp. which employs probes specific for 23S rRNA. The detection of S. aureus in biological samples is important as within the genus Staphylococcus, S. aureus is the most clinically significant species due to the incidence and severity of the infections it can cause (Morse, Staphylococci, in Medical Microbiology and Infectious Diseases, Abraham Braude, editor, W. B. Saunders Company, Philadelphia, Pa., 1981). Moreover, S. aureus is a prominent agent of nosocomial infections, and methicillin resistant strains (MRSA) have emerged as a major epidemiological problem in hospitals throughout the United States.
Vancomycin resistant enterococci (VRE) represent another emerging class of drug resistant bacteria. Since these organisms were first identified in 1986, nearly 30 years after vancomycin was clinically introduced, it has been established that vancomycin resistance is primarily conferred by either of two functionally similar operons, VanA and VanB. These operons, transfer of which can be mediated by plasmids or transposons, are complex resistance determinants that may have evolved in other species and then been acquired by enterococci. Frequently identified risk factors for VRE colonization and infection include prolonged hospital stays, exposure to intensive care units, transplants, hematologic malignancies, and exposure to antibiotics. Notably, more than 95% of VRE recovered in the United States are Enterococcus faecium, and virtually all are resistant to high levels of ampicillin. (Rice, L., Emerging Infectious Diseases 7:183 (2001)) Because vancomycin resistance is transferrable by genetic means, there is the possibility that resistance to this important antibiotic can be acquired by other microorganisms, such as Staphylococcus aureus. Indeed, conjugative transfer of the VanA gene from Enterococcus faecalis to S. aureus has already been demonstrated in vitro. It has been speculated that such a transfer mechanism may underlie the appearance of vancomycin resistant S. aureus (VRSA). (Noble et al., FEMS Microbiol Lett 93:195 (1992)) Conceivably, there could emerge a strain of S. aureus which has acquired resistance to both methicillin and vancomycin (MVSA).
Accordingly, there is a continuing need for the rapid processing of clinical or biological samples, and for the rapid and accurate detection of pathogens and antibiotic resistance genes in clinical samples.